Various biologic products are produced from biological materials, such as blood plasma, obtained from human and non-human sources. The various fractions of protein materials separated from human plasma that are useful as biologic products can include albumin, antithrombin III, Factor VIII (anti-hemophilic factor [AHF], and the prothrombin complex (PTC) which includes Factor IX, together with Factors II, VII and X.
Factor IX participates in the cascade of events that leads to blood coagulation. Factor IX is absent or deficient in patients who have a condition identified as "Hemophilia B". Thus, the blood of Hemophilia B patients does not clot properly. Factor IX is administered to Hemophilia B patients to provide sufficient Factor IX to return the clotting ability of their blood to as close to normal as possible.
The Factor IX concentrates that are presently available include other blood factors in addition to Factor IX. For example, the prothrombin complex mentioned above includes Factors II, VII and X in addition to Factor IX.
The occurence of thrombotic complications, such as deep vein thrombosis, disseminated intravascular coagulation (DIC) and pulmonary embolism has been reported in patients treated with prothrombin complex concentrates. These complications are frequently seen in premature infants, patients with poor liver function and surgery patients. They have also been observed in Hemophilia A patients receiving prothrombin complex concentrate as a Factor VIII inhibitor bypassing agent.
The thrombogenic component of prothrombin complex concentrates has most often been attributed to either activated Factors, coagulant active phospholipid or zymogen overload. Zymogen overload may be the basis of DIC in surgical situations where patients receive large and repetitive doses of prothrombin complex concentrates. In such cases, a buildup of zymogens in the circulation, particularly of prothrombin and Factor X, is likely to occur due to their relatively long half-life in relation to Factor IX.
Because of the thrombotic complications associated with the use of prothrombin complex, it is desirable to provide a Factor IX concentrate essentially free of other proteins for use in treating Hemophilia B patients.
In the human blood clotting cascade, Factor X in its active form (Factor Xa) cleaves prothrombin (Factor II) to form thrombin (Factor IIa). Thrombin then acts on fibrinogen to form fibrin which becomes the structural protein of the clot. Providing an external source of purified human Factor X or Xa may be desirable in the treatment of certain patients with bleeding disorders related to Factor X or other coagulation Factor deficiencies.
Various methods for enhancing the purity of Factor IX and/or Factor X concentrates have been reported. For example, processes for producing concentrates of Factor IX essentially free of prothrombin, Factor VII and Factor X by use of affinity chromatography on a sulfated dextran gel have been disclosed (D. Menache et al, "Coagulation Factor IX Concentrate: Method of Preparation and Assessment of Potential In Vivo Thrombogenicity in Animal Models", Blood, Vol. 64, No. 6, December, 1984, pp. 1220-1227). Factor IX has also been disclosed as having been purified by affinity chromatography on a heparin-Sepharose (agarose) gel (L-O. Andersson et al, "Purification and Characterization of Human Factor IX", Thrombosis Research, Vol. 7, 1975, pp. 451-459). Factor IX and Factor X have been separated by using a process which includes heparin-agarose chromatographic techniques (S. P. Bajaj et al, "A Simplified Procedure For Purification of Human Prothrombin Factor IX and Factor X", Preparative Biochemistry, 11(4), 1981, pp. 397-412). Procedures are also known in the art for separating Factor IX by affinity chromatography on a dextran sulfate-sepharose (agarose) gel.
While Factors IX and X can be separated on agarose gels in the laboratory, the use of agarose gels for large scale separations has been found to be unsatisfactory. When the agarose gels are put into commercial size columns, they compress to an undesirable extent and thereby inhibit flow of liquids through the column.
It is, therefore, desirable that a process for separation of Factor IX and/or Factor X from other proteins be provided that utilizes a chromatographic resin that has sufficient rigidity so that it can be used in large commercial size columns without causing a excessive pressure drop.